J Control Release 2014 Jan 10;173:96-101. doi: 10.1016/j.jconrel.2013.10.036.

Nano-visualization of viral DNA breaching the nucleocytoplasmic barrier.

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Nuclear pore complexes (NPCs) mediate all transport between the cytosol and the nucleus highly selectively. Their selectivity can become an insurmountable hurdle for exogenously applied therapeutic macromolecules. Many viruses naturally overcome the NPC barrier. Therefore, gene therapy often utilizes viral particles as nano-carriers for exogenous therapeutic macromolecules. Viral gene therapy, however, frequently leads to severe adverse effects. We intend to elucidate the mechanisms underlying controlled release of viral DNA at the NPC in order to design new non-viral approach for intranuclear DNA delivery. For this purpose, we developed a comprehensive experimental strategy combining nano-imaging and biochemical methods. Here, we apply Herpes simplex virus type 1 (HSV-1) as an ideal example. HSV-1 contains its long 145kbp DNA in a capsid which is merely 125nm in size. The capsid shields and targets the DNA specifically to the NPC. Only at the NPC, the capsid releases the DNA for nuclear delivery. The underlying mechanisms of this multi-step process remain unresolved. In this work we follow the fate of HSV-1 DNA in the process of transit across the NPC. Our results indicate an involvement of hydrophobicity for capsid opening. Furthermore, the DNA is presumably released as a single thread. We assume that it penetrates the NPC in this conformation. It is compacted by the host intranuclear proteins once it reaches the interior of the nucleus. Our proposed experimental strategy can be extended to other viruses. Moreover, our observations may help design potent non-viral based nano-carriers for gene therapy.

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